The Rhizobium - legume symbiosis is a good model system for the genetic analysis of multicellular development, since the bacterial and plant genomes can be altered independently. During symbiosis, a cooperative interaction between bacterial and plant cells results in the formation of a specialized plant organ called a root nodule, within which the rhizobia fix nitrogen. In this proposal, an experimental approach is presented for the analysis of specific interactions during the symbiosis of Rhizobium meliloti and its leguminous host, Medicago sativa (alfalfa). The long term goal will be to identify key biochemical pathways of R. meliloti which may be required for the transmission of signals which are involved in the communication of the bacteria with plant. The development of ineffective nodules induced by mutant strains of R. meliloti will be analyzed by a previously devised immunoassay of nodule specific alfalfa proteins or nodulins. R. meliloti strains will be identified which induce nodules whose development is arrested at the same step. Such bacteria should have related biochemical defects which prevent at most, a few particular bacterial-plant interactions. To identify strains which are defective in a specific bacterial-plant interaction, functional complementation tests will be performed. Nodules induced by a mixture of two R. meliloti mutant strains will be compared to nodules induced by each of the individual strains. Those R. meliloti mutant strains that disrupt independent bacterial plant interactions should complement, at least partially, resulting in changes in the accumulation of nodulins by the alfalfa host. The strains within one such functional complementation group should have related biochemical defects which prevent a particular bacterial-plant interaction. The genes defined by the mutations in R. meliloti strains within one functional complementation group will be isolated and cloned. The biochemical function of the product of each cloned gene will be investigated by analyzing the location, the transcriptional properties, and the DNA sequence of the gene. We will then search for DNA or protein sequence homology with genes of known function. Results will provide clues to the biochemical functions of the cloned genes. These clues may enable us, to test the importance of specific R. meliloti biochemical pathways that may be required for the interaction of Rhizobium with alfalfa.